Major histocompatibility complex (MHC) class I molecules bind to peptide antigens and present these antigens to CD8 T cells. Human MHC class I variants are encoded by the HLA-A, HLA-B and HLA-C genes. These genes are highly polymorphic. The polymorphisms influence outcomes in a number of infectious diseases, cancers and inflammatory diseases. Among the three HLA class I loci, HLA-B molecules are shown have dominant influences upon outcomes in multiple diseases. A given HLA class I allotype can bind to a large number of cellular and pathogen-derived peptides, collectively called the peptidome. The diversity of HLA-B peptidomes could be one factor underlying their different effects upon disease outcomes. A number of factors are predicted to influence peptidome diversity among HLA class I allotypes, including the structure of the peptide-binding site, the intracellular assembly mechanism and the intrinsic stability of the peptide-deficient form. Several HLA-B molecules are assembled via unconventional intracellular pathways that are suggestive of the presence of novel unconventional epitopes within their peptidomes. We propose to use liquid chromatography mass spectrometry (LC-MS) methods to quantify, characterize and understand the full breadth of the peptidomes of selected HLA-B variants. Based on existing MS datasets, we will also develop globally normalized methods to quantify and compare peptidome diversities of different HLA-B variants. The findings of this study will be significant towards epitope and HLA selection during vaccine design against cancers and infectious diseases. The studies will also establish methods to identify novel disease-relevant epitopes, and allow a better understanding of the relationships between HLA class I genotypes and disease outcomes.